Azithromycin Prevents Subglottic Stenosis in Mice

Abstract

Objective

Pediatric subglottic stenosis (SGS) is characterized by subglottic narrowing which occurs when pathological fibroblasts deposit extracellular matrix that reduces airway patency. Recent clinical observations have suggested that azithromycin may have favorable impacts on SGS reduction while treating airway infections; furthermore, our recent work in mice demonstrated that the airway microbiome influences SGS. In this work, we characterize the protective effect of azithromycin as an immunomodulatory and antibacterial therapeutic against subglottic stenosis.

Methods

Immunomodulatory and antifibrotic effects of azithromycin were assessed on TGF‐β1‐stimulated airway fibroblasts at 10 μg/mL for 5 days. Changes in gene expression were quantified by RT‐qPCR and myofibroblast differentiation by α‐SMA immunostaining. Murine airways were pretreated (2‐weeks) with intranasal azithromycin before SGS injury by a twisted wire brush. Disease severity and immune response were characterized by histology and immunostaining for immune cells.

Results

In vitro, azithromycin treatment of TGF‐β1‐stimulated fibroblasts exhibited strong reductions in extracellular matrix (COL1A1, LOX) and myofibroblast‐related gene expression (ACTA2). Notably, there was a significant reduction in pro‐fibrotic expression, which was observed with 10 μg/mL azithromycin. Immunostaining of fibroblasts for α‐SMA revealed strong reductions in the number of positive‐staining cells and the intensity of each positive cell. In vivo, azithromycin exhibited a significant decrease in lamina propria thickness indicative of reduced stenosis with associated changes in T‐cell infiltration.

Conclusions

Overall, we show azithromycin prevents pro‐fibrotic gene expression and myofibroblast differentiation and can help protect mice from developing SGS. This introduces azithromycin as a potential treatment for SGS.

Level of Evidence

NA Laryngoscope, 135:409–415, 2025

Azithromycin is a frequently prescribed antibiotic to pediatric patients with subglottic stenosis; however, the indication for this administration is infection prevention or resolution. In this work, we show that azithromycin not only has strong antifibrotic activity but also a protective effect against subglottic stenosis development in mice.

INTRODUCTION

Pediatric subglottic stenosis (SGS) is the narrowing of the subglottis and scarring of the vocal folds by fibrosis, often arising post‐intubation in infants and children. ¹ SGS is an inflammatory and fibrotic disease by nature and in its iatrogenic form is precipitated by an inflammatory cascade following injury of the airway mucosa and lamina propria. ² This pathway is crucially mediated by key immune cell populations such as T cells, which invade the lamina propria and release chemokines and pro‐inflammatory cytokines, particularly TNFα, IL‐1β, IL‐6, and IL17A. ³ , ⁴ , ⁵ These inflammatory signals in turn trigger fibrosis by promoting myofibroblast differentiation while also modulating extracellular matrix (ECM) protein accumulation and matrix metalloproteinase secretion. ² , ⁶ , ⁷ In addition to these inflammatory and fibrotic pathways, recent studies have indicated a link between airway microbiome composition and SGS. Specifically, adult SGS patients have been shown to exhibit bacterial profiles in the airway that are distinctly different from controls, characterized by dysregulated levels of multiple genera including Prevotella and Streptococcus. ⁸ Furthermore, we have previously shown that the local administration of antimicrobial peptides via a drug‐eluting platform mitigated SGS in mice therefore alluding to the connection between the airway microbiota and SGS. ⁹ Still, there exist no specific treatments for SGS that exploit this bacterial‐host interaction.

Current treatments of SGS depending on the grade or severity of the stenosis range from less invasive surgical approaches such as endoscopic lysis of scar tissue and balloon dilation to more invasive options such as laryngotracheal reconstruction and cricotracheal resection. ¹⁰ , ¹¹ However, these treatments can fail and may require revision surgery to treat restenosis. ¹² , ¹³ Thus, a preventative treatment to avoid the development of SGS would be ideal.

Pediatric patients with SGS are often prescribed azithromycin (AZM)—an antibiotic of the macrolide family—which is known to have both antibacterial and immunomodulatory properties. ¹⁴ , ¹⁵ Thus, it holds the potential to regulate both the imbalanced airway microbiome and the inflammatory cascade associated with SGS. Clinical observations and case studies have suggested that SGS patients treated with AZM exhibited reduced stenosis and improvement of symptoms, ¹⁶ , ¹⁷ raising the question of whether the drug could be used as an anti‐stenotic therapeutic.

Our goal in this work was to investigate AZM as a therapeutic for SGS. We hypothesize that AZM can reduce SGS and does so by influencing transcriptional activity of key genes mediating inflammation and fibrosis. To test our hypotheses, we cultured human airway fibroblasts with pro‐fibrotic cytokine and treated them with AZM, assessing differential transcriptional activity and myofibrotic protein expression. Furthermore, we administered azithromycin in a validated murine model of SGS to investigate its efficacy in reducing stenosis and stenosis‐inducing immune pathways in vivo.

METHODS

In Vitro Azithromycin Treatment

Human airway fibroblasts (hAFs) were cultured in growth medium (DMEM, 10% FBS, 2% PSF, 1X MEM NEAA) at 37°C, 5% CO₂, and 95% humidity. Cells were seeded in a 6‐well plate at 15,000 cells/well and allowed to adhere overnight and then treated with the addition of 10 μg/mL azithromycin dihydrate or without in treatment medium (DMEM, 2.5% FBS, 2% PSF, 1X MEM NEAA) for 1 day. They were then stimulated with 10 ng/mL TGF‐β1 (PeproTech) in addition to azithromycin dihydrate (Tokyo Chemical Industry Co.) or without for 5 additional days and compared with untreated controls.

RT‐qPCR

Cells were lysed and RNA extracted following manufacturer protocols for the RNeasy Plus Mini Kit (Qiagen). cDNA was synthesized with SuperScript IV First‐Strand Synthesis System (Invitrogen) with random hexamer primers. Quantitative real‐time PCR was performed with SYBR® Green Master Mix (Applied Biosystems) on a Quantstudio 7. Fold change was calculated as 2^−ΔΔCT and normalizing to 18S and Day 0 gene amplification. Primers are listed in Supplementary Table 1.

Immunocytochemistry

Cells were fixed in 4% paraformaldehyde for 15 min, blocked in blocking buffer (PBS, 5% normal goat serum [Cell Signaling], 0.3% Triton‐X100), and stained with a mouse anti‐human α‐SMA primary antibody (Invitrogen, clone 1A4) and an anti‐mouse IgG (H + L), F(ab’)₂ Fragment secondary antibody (Cell Signaling, Alexa Fluor 647), phalloidin (Invitrogen), and DAPI (Invitrogen). Slides were then mounted in Fluoramount‐G and imaged with a BZ‐X810 All‐in‐One Fluorescent Microscope (Keyence, IL).

In Vivo SGS Model

Male C57BL/6 mice 8 weeks of age were housed in specific pathogen‐free conditions in accordance with IACUC 1356 at the Children's Hospital of Philadelphia. Mice were randomly assigned to obtain n = 3 per group of the following: (1) control/sham, (2) control/SGS, (3) azithromycin/sham, and (4) azithromycin/SGS. Mice were intranasally administered 50 μL of water with 1 g/L sucralose for the control conditions or 1 g/L azithromycin for the azithromycin conditions supplemented with 1 g/L sucralose weekly for 2 weeks. Mice were allowed to drink these same solutions for each condition in their drinking water ad libitum. SGS was then induced following previously established protocols. ¹⁸ Briefly, mice were sedated with isoflurane and intubated with a 22‐gage blunt needle. Airways were brushed 15 times with a 0.02″ diameter twisted wire brush (McMaster Carr) dipped in 1 U/mL bleomycin. Mice continued their treatments until euthanasia at either 7 or 21 days.

Lamina propria and epithelium thicknesses were quantified by averaging the respective thicknesses measured at 4 locations around the tracheal section to account for circumferential differences in thickness.

Immunofluorescence Staining

Excised mouse tracheas were fixed in 10% buffered formalin for 1 h, embedded in paraffin, and sectioned at 5 μm. For immunofluorescence staining, antigen retrieval was conducted with 1:1000 proteinase K (Qiagen) for 30 min at room temperature, then blocked for 2 h with blocking buffer (PBS, 5% goat serum, 0.3% Triton X‐100). Sections were treated with primary antibody rabbit anti‐mouse CD3ε (Cell Signaling 78588) 1:400 at 4°C. Anti‐rabbit IgG F(ab’)2 Fragment (Cell Signaling 4412) 1:1000 at room temperature for 2 h with DAPI (Invitrogen). Lastly, samples were mounted and imaged with a BZ‐X810 All‐in‐One Fluorescent Microscope (Keyence). T‐cell infiltration was quantified by counting the number of CD3ε‐positive cells present in the imaged samples.

Statistical Analysis

Data were analyzed and reported as mean +/− standard deviation with replicate and 95% confidence intervals. Ordinary one‐way analysis of variance (ANOVA) with Tukey's post hoc multiple comparisons was used to compare gene expression, while a two‐way ANOVA with Tukey's post hoc multiple comparisons was used for histology thicknesses and T‐cell counts. Lamina propria and epithelial thicknesses were measured with FIJI (FIJI Is Just ImageJ) v1.54h. Data analyses were performed using GraphPad Prism Software Version 10.0.0.

RESULTS

Azithromycin Reduces Pro‐Inflammatory and Pro‐Fibrotic Fibroblast Phenotypes In Vitro

Human airway fibroblasts (hAFs) were stimulated with 10 ng/mL of TGF‐β1 and subsequently stimulated with azithromycin sulfate (AZM) for 5 days. Gene expression was compared against untreated cells and TGF‐β1 only treated controls. TGF‐β1 stimulation significantly increased the expression of the pro‐inflammatory cytokine IL6 from 2.716 (n = 6, 95% CI: 0.918–4.514) to 5.596‐fold (n = 6, 95% CI: 4.123–7.069, p < 0.01), which was returned to 2.639‐fold (n = 6, 95% CI: 0.2.199–3.079, p < 0.01) with AZM treatment (Fig. 1, upper left). The expression of ACTA2 increased from 0.281 (n = 6, 95% CI: 0.238–0.324) to 2.576‐fold (n = 6, 95% CI: 1.708–3.443, p < 0.0001) (Fig. 1, upper right). AZM returned the ACTA2 expression to 0.550‐fold (n = 6, 95% CI: 0.455–0.646, p < 0.0001), which was similar to the expression of unstimulated cells. Extracellular matrix component COL1A1 was significantly increased to 2.253‐fold (n = 6, 95% CI: 1.855–2.651) from 0.3433 (n = 6, 95% CI: 0.298–0.389), which with AZM decreased significantly to 1.702 (n = 6, 95% CI: 1.340–2.064, p < 0.05) (Fig. 1, lower left). This level remained significantly greater than the untreated control (p < 0.0001). Matrix crosslinking enzyme LOX expression increased from 1.230‐fold (n = 6, 95% CI: 0.985–1.475) to 2.642 (n = 6, 95% CI: 1.975–3.309, p < 0.0001), which decreased significantly to a similar expression of 1.175 (n = 6, 95% CI: 1.010–1.339, p < 0.0001) to the untreated control (Fig. 1, lower right).

Fig. 1.

Gene expression of the pro‐inflammatory cytokine IL6, myofibroblast marker ACTA2, extracellular matrix component COL1A1, and extracellular matrix crosslinking enzyme LOX by RT‐qPCR. N = 5; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Azithromycin Reduced α‐SMA ⁺ Fibroblasts

hAFs stimulated and treated in the same manner as for gene expression were immunostained with alpha smooth muscle actin (α‐SMA), and co‐stained with for F‐actin and DAPI (Fig. 2). Untreated control cells exhibited minimum α‐SMA⁺ cells, while TGF‐β1 stimulation greatly increased α‐SMA⁺ cell presence. AZM treatment reduced the presence of α‐SMA⁺ cells and the intensity of staining per cell.

Fig. 2.

Immunostaining of untreated control, TGF‐β1‐stimulated, and TGF‐β1‐stimulated AZM‐treated hAFs for α‐SMA (magenta), cell nuclei (cyan), and F‐actin (white). Scale bar = 200 μm.

Azithromycin Reduces Airway Thickness In Vivo

Mice were pretreated with either drinking water with sucralose or AZM with sucralose solution for 2 weeks and then intubated and brushed with a twisted wire brush dipped in bleomycin to induce SGS. Histology of control mice revealed thicker lamina propria and epithelium at day 7 and a thicker lamina propria but thinner epithelium at day 21 compared with uninjured shams (Fig. 3A–C, Supplementary Figure 1). The epithelial layer of azithromycin‐treated SGS mouse tracheas were observably thicker than sham controls at day 7 but appeared less thick at day 21 than day 7 (Fig. 3D–F, Supplementary Figure 2). Subsequent quantification, however, showed no statistical difference between these three thicknesses (Fig. 4).

Fig. 3.

Representative histological staining by hematoxylin and eosin of control mice (A) uninjured sham, (B) 7 days, and (C) 21 days after subglottic stenosis (SGS) injury. Azithromycin‐treated mice (D) uninjured sham, (E) 7 days, and (F) 21 days after SGS injury. N = 3, biological replicates for each condition and treatment. 40× magnification scale bar = 50 μm, 10× magnification inset scale bar = 300 μm.

Fig. 4.

Quantification of (A) lamina propria and (B) epithelium thickness between the vocal folds and cricoid for uninjured sham, 7, and 21 days after subglottic stenosis injury with and without azithromycin treatment. N = 3, biological replicates for each condition and treatment.

Thicknesses were subsequently quantified revealing sham airways averaging a mean thickness of 14.50 μm (n = 3, 95% CI: 6.856–22.15), which significantly increased to 42.32 μm (n = 3, 95% CI: 23.18–61.46, p < 0.0001) after 7 days and to 45.31 μm (n = 3, 95% CI: 32.28–58.33, p < 0.0001) after 21 days. Conversely, AZM‐treated mice exhibited no statistically significant change in lamina propria thickness over the course of all 21 days compared with sham controls.

Epithelium thickness was also measured and revealed increased thickening after 7 days from 20.40 μm (n = 3, 95% CI: 16.73–24.07) to 25.97 μm (n = 3, 95% CI: 19.13–32.81, p < 0.01) and significant thinning after 21 days to 14.32 μm (n = 3, 95% CI: 12.34–16.30) compared with uninjured sham airways (p < 0.01) and day 7 post‐injury (p < 0.0001). AZM‐treated mice exhibited no significant change in epithelial thickness over 21 days after injury.

Azithromycin Treatment Mitigates T‐Cell Infiltration

Mouse airways were immunostained for anti‐CD3 to quantify T‐cell infiltration (Fig. 5). In control mouse airways, T‐cell infiltration increased from an average of 10.67 T cells (n = 3, 95% CI: 6.87–14.46) to 27.67 (n = 3, 95% CI: 5.40–49.93, p < 0.05) after 7 days and 67.67 (n = 3, 95% CI: 35.34–99.99, p < 0.0001) after 21 days post‐injury. In AZM‐treated mice, there were similar T‐cell counts between sham, 7 day post‐injury, and 21 day post‐injury.

Fig. 5.

CD3+ T cells stained from mouse tracheal sections from control mice before and after injury and azithromycin‐treated mice before and after injury. T‐cell quantification of 3 biological replicates. 40× magnification scale bar = 50 μm; *p < 0.05; ****p < 0.0001.

DISCUSSION

In this work, we demonstrated that treatment with the widely used macrolide antibiotic, azithromycin (AZM), protects mice from SGS through its anti‐inflammatory and antifibrotic activity directed toward airway fibroblasts. To assess this, we first tested the effect of AZM on cultured human airway fibroblasts (hAFs) with TGF‐β1—a pro‐fibrotic and immunomodulatory cytokine implicated in fibrotic diseases—to promote a pro‐fibrotic phenotype, then treated with 10 μg/ML of AZM for 5 days before assessing gene expression. Immunostaining was conducted for further confirmation. We then assessed the efficacy of AZM in reducing SGS in our well‐established mouse model of SGS. ¹⁸ In this approach, we pretreated mice with AZM prior to disease induction to assess the potential as a preventative treatment. Histological analyses and immunostaining were conducted to verify disease pathology and progression.

AZM treatment was successful in diminishing pro‐inflammatory and pro‐fibrotic phenotypes in hAFs as evidenced by significantly diminished transcription of pro‐inflammatory cytokine IL6, of myofibroblast marker ACTA2, of ECM protein COL1A1, and of the collagen crosslinking enzyme LOX, suggesting AZM reduces the inflammatory and fibrotic drivers of SGS via transcriptional control. This has been previously shown in other fibroblasts in vitro from the lower airway of humans and mice in the treatment of idiopathic pulmonary fibrosis. ¹⁹ , ²⁰ Furthermore, immunostaining corroborated the decreased expression of alpha–smooth muscle actin (α‐SMA) in treated cells, as well as the reduced number of cells expressing α‐SMA. Furthermore, there appeared to be a qualitative decrease in the intensity of α‐SMA in the cells that stained positive. The exact pathways by which AZM downregulates transcriptional activity of these genes are poorly understood and require further investigation; however, some there are a few studies that suggest numerous pathways. One study showed that AZM can modulate the NFkB pathway lung inflammation in vivo, ²¹ raising the question whether interference with this pathway could be implicated in AZM's stenosis‐reducing activity as well. Another study hypothesized that AZM regulates pro‐apoptotic effects in airway fibroblasts, which could explain the change in phenotype and eventual disease regression. Other groups discuss the potential influence of AZM on cellular autophagy, where myofibroblast differentiation is reduced through the proteasomal degradation of NOX4 which regulates autophagy in these cell types. ²²

In our in vivo experiments, mice pretreated with AZM demonstrated no significant difference in epithelium and lamina propria thickness compared with sham controls at 7 days post‐injury and continuing 21 days, while those not treated with AZM had significantly greater thicknesses in both layers at day 7. Interestingly, non‐treated SGS mice had significantly thicker epithelia but thinner lamina propria than sham controls at day 21. Regardless, AZM's efficacy in maintaining consistently healthy airway thickness in SGS mice over a prolonged period of 3 weeks demonstrates its viability as a stenosis‐reducing therapeutic.

In previous work, we and others have suggested that bacteria play a crucial role in the development of SGS and when attenuated with antimicrobial agents, SGS can be reduced. ⁸ , ⁹ , ²³ Therefore, we are unsure whether the activity of AZM is strictly through modulation of fibroblastic pathways or also mediating the bacterial populations in the airway as well. Some groups suggest that AZM modulates the activity of inflammatory and fibrotic pathways through various cell types beyond fibroblasts alone. For example, one study indicates that AZM downregulates certain inflammatory genes including MUC5AC and MMP9 in airway epithelial cells. ²⁴ Another study shows how AZM shifts macrophages toward the alternatively activated phenotype, leading to altered fibrotic activity when fibroblasts are co‐cultured in the presence of P. aeruginosa. ²⁵ Yet, another study demonstrates how AZM suppresses epithelial mesenchymal transition (EMT), a process in which epithelial cells can acquire myofibroblast phenotypes upon activation by signals such as TGF‐β. ²⁶ , ²⁷ EMT has been implicated as a major driver of idiopathic subglottic stenosis; therefore, AZM's direct effects on epithelial cells could explain the regulation of epithelial thickness in treated mice in our study. ⁴ , ²⁸ Considering our findings and those in the existing literature, macrolide suppression of stenosis most likely entails a much more complex nexus of cellular and bacterial mechanisms than direct effects on fibroblasts, and more comprehensive studies are needed to help elucidate these. Nonetheless, our findings suggest AZM as a robust preventative treatment for SGS as it reduces airway fibrosis and subsequent stenosis.

Furthermore, AZM was successful in modulating immune cell infiltration in SGS mice tracheas. AZM‐treated mice with induced SGS exhibited similar numbers of invading T cells at the site of stenosis compared with sham controls throughout 21 days post‐injury. Non‐treated SGS mice, however, showed significantly higher, progressively increasing T‐cell counts from day 7 to day 21 compared with sham controls. T cells have been implicated in the pathogenesis of SGS through their role in the IL‐17A/IL‐23 cytokine signaling pathway, which promotes fibroblast proliferation and ECM deposition. ⁵ , ²⁹ AZM's efficacy in suppressing these key pathological mediators further corroborates its robustness in mitigating SGS. Future studies examining how AZM might interact with the IL‐17A/IL‐23 pathway and other T‐cell signaling pathways can help further elucidate the therapeutic mechanisms of this drug.

Overall, our experimental observations that AZM diminishes airway stenosis and immune cell infiltration in vivo align with previous clinical observations that macrolide treatment can mitigate SGS in human patients. ¹⁶ , ¹⁷ Furthermore, the transcriptional and phenotypic effects observed in vitro provide plausible insight into the mechanisms by which AZM could accomplish these effects beyond its antimicrobial activity. We note that our study does present some limitations. We do not specifically investigate the contribution of AZM's antimicrobial activity in its fibrosis and stenosis‐reducing effects. Furthermore, our in vivo experiments do not clearly distinguish specific cell‐type effects on the reduction of airway thickness and immune cell recruitment. Future studies will include cellular phenotyping to investigate which cells are responding to injury and altering their behavior with AZM treatment at early and late time points to capture any time‐dependent mechanisms. Additionally, the method of nasal AZM administration in vivo and the use of chemomechanical injury to simulate SGS may not correlate exactly with treatment of human SGS. Still, the use of AZM to treat SGS is appealing compared with current surgical treatment methods in that it is noninvasive and would not require surgical follow‐ups. Additionally, AZM could be used as an adjuvant in combination with current treatments to increase the efficacy of treatment and address varying severities of SGS. AZM treatment does, however, present potential drawbacks, especially with it being an antibiotic that can cause bacterial resistance over time, as well as adverse off‐target symptoms upon systemic and long‐term administration such as gastrointestinal distress and neurodegenerative effects including paresthesia and hearing loss. ³⁰ , ³¹ Optimization of dosage regimens and employment of targeted drug delivery methods have been promising for other therapeutics, such as steroids and peptides on drug‐eluting endotracheal tubes or stents, ⁹ , ³² which could act as a good strategy to help surmount these issues.

CONCLUSION

We demonstrated how AZM downregulates pro‐inflammatory and pro‐fibrotic phenotypes via transcriptional modulation in vitro. Furthermore, we demonstrate the efficacy of AZM in reducing stenosis and mitigating T‐cell infiltration in a murine model of SGS. Overall, AZM presents itself as a promising therapeutic drug for prevention of SGS. Further investigation is required before clinical translation.

Supporting information

Supplementary Table 1 RT‐qPCR Primers S1.

Supplementary Figure 1. Low magnification images of control airway sections. N = 3. Scale bars = 200 μm.

Supplementary Figure 2. Low magnification images of azithromycin airway sections. N = 3. Scale bars = 200 μm.